Signaling pathways employing heterotrimeric guanine nucleotide-binding proteins (G proteins) and mitogen-activated protein kinases (MAP kinase or MAPK) allow mammalian cells to sense light and olfactants, regulate synaptic transmission, control cardiac rate and force, modulate blood clotting, regulate synaptic transmission, control cardiac rate and force, modulate blood clotting, regulate smooth muscle contraction , and control cell proliferation and differentiation. Mutant forms of G proteins, their receptors, and downstream effectors cause various diseases in human, including endocrine disorders, retinal degeneration, and pituitary, thyroid, adrenal cortical and ovarian tumors. These signaling pathways therefore provide important pharmacological targets for controlling human diseases. This project focuses on the mechanisms whereby; 1) agonists activate their cognate G protein-coupled receptor; and 2) G protein betagammma subunits activate a specific MAPK pathway. The mating pheromone response pathway of the yeast Saccharomyces cerevisiae is used as a model that offers genetic and biochemical tools unavailable in mammalian systems. In the short-term, six specific aims will be addressed; 1) define receptor domains that govern the activation process by generating and characterizing an extensive collection of constitutively active receptor mutants; 2) determine whether mutant receptors that cause constitutive signaling in vivo are constitutively active in vitro in a purified reconstituted system; 3) determine whether Gbetagamma subunits or members of the rho family of small GTP-binding proteins bind and/or activate Ste20p protein kinase in vitro; 4) determine whether Gbetagamma subunits target Ste20p to the plasma membrane, and whether constitutive targeting of Ste20p to the membrane bypasses the requirement for Gbetagamma subunits; 5) define domains of Ste20p that are important for regulation by Gbetagamma subunits and/or rho family members; 6) if Ste20p is not the direct target of Gbetagamma subunits, then direct targets will be identified by using Gbetagamma affinity columns, isolating Gbetagamma-effector complexes, or using two- hybrid screens. Given the extensive similarities between G protein- and MAP kinase-lined signaling pathways in yeast and mammalian cells, these studies will reveal fundamental mechanisms governing signaling by receptors and Gbetagamma subunits in mammalian cells.